Sexual hormones and method of preparing them



Patented Apr. 23, 1940 SEXUAL HORMONES AND METHOD OF PREPARING THEMWilhelm Dirscherl, Frankfort-on-the-Main, and Fritz Hanusch, Heidelberg,Germannassig'nors to Bare Chemicals Inc., Nepera Park, N. Y., acorporation oi New York No Drawing. Application November 17, 1936,Serial No. 111,330. In Germany November 18,

Our invention relates to substances acting as sexual hormones andespecially to substances having the activity of a male sexual hormone aswell as of a corpus luteum hormone. It deals especially with the mannerof preparing such substances in a new and a more efiicient way than itwas possible up till now. Other objects will hereinafter appear.

As it is well known to those skilled in the art,

there have been attempts to oxidize cholestenone by'chromic acid in anacid medium. Inasmuch as these experiments were carried through in thecold they did not succeed'at all; on working in the warmth it was statedthat after a consumption of 6 atoms oxygen 50-60% cholestenone wereunattacked. No oxidation products whatever have been isolated.

We have found that hormone like acting substances can be obtained bytreating cholestenone in the acid or neutral medium by oxidizing agents.The substances are prepared by separating after the oxidation thereacting mixture from inefllcient, acid and volatile components and fromunchanged initial material and by concentrating, purifying and isolatingin the manner usual in preparing sexual hormones.

As initial materials are taken into account the pure cholestenone,obtained for instance from cholesterol by halogenation, oxidation anddehalogenation or mixtures containing this substance, obtained, e. g.,from wool fat in an analogous manner.

Oxidation may be carried out by oxidizing agents especially vigorouslyacting oxidizing agents, particularly by metal compounds rich in oxygenas, e. g., chromic acid, also called chromic anhydride, permanganicacid; acetate of tetravalent lead Pb(OCOCHa)4, further by acids whichare derived from hydrogen peroxide as, e. g., peracetic acid.

Concentrated nitric acid is not suitable as an oxidizing agent. Thereaction is carried out preferably in the presence of diluentsespecially of organic character, as, e. g., glacial acetic acid orstrong acetic acid. Oxidation by electrolysis may also be taken intoaccount whereby the above named metal compounds can act as oxygentransporters.

Oxidation may be executed at room temperature as well as at highertemperatures, preferably at temperatures between 30.- C., temperaturesbetween 70-100 0., and by special conditions e. g. in case of a suitabledilution or a very-short reaction time, temperatures above so 100' C.may also be used. It is recommended to 13 Claims. (Cl. 260-397) takecare of a good thorough mixing of the oxidizing agent with the reactionmixture containing the substance to be oxidized which can take place bystirring or eventually by adding emulsifying substances.

After having finshed the oxidation an excess of the oxidant, if any, isdestroyed by'means of easily oxidizable substances, as for examplemethanol, formic aldehyde, sulphurous acid and The solvent is nowremoved by distillation for instance, after neutralising the mixture,preferably in vacuo. The remaining mixture of substances is separatedfrom inorganic components by means of water, whilst the organic compo-a.

inactive volatile components are distilled in vacuo or with a suitablevapor as a carrier as for instance water vapor. In some cases thesequence of removing the acid and volatile reaction components mayalsobe reversed.

The product'thus obtained, a brownish oil, shows a strong male hormonelike activity and at the same time the activity of a corpus luteumhormone.

For further purification, still existing inactive substances, especiallyunattacked cholestenone', are separated from the active substances. Thiscan be carried out preferably by the following ways:

1. By separation by means of fractional crystallisation from suitablesolvents preferably at low temperatures; 2. By treating with adsorptionagents as, e. g., alumina or bleaching clay; by acting in this mannerthe hormone acting substance remains as a rule fixed on the adsorptionagent whilst the initial material, the cholestenone, is substantiallyremoved without any diiliculty by a thorough washing; v

3. By distributing amongst two solvents as, e. g., ligroin and anaqueous alcohol; I

4. By treating with ketone reagents as, e. g., hydroxylamine,hydrazines, especially simple or substituted phenyl hydrazines,semicarbazide, derivatives of aminoacethydrazide and so on, the additioncompounds thus formed being separated by one of the methods 1-3.

. lly commendable are combinationsoif the said methods particularly thecombination' oi fractional crystallisation. together follow;-

s a rption.

being enriched by the substance showing ,the activity of a male hormoneand the other by the substance showing the activity of a corpus luteumhormone and. iinally a separation rather: complete'oi the two hormonesubstances may attained. l g v Instead of concentrating the activelretone's "themselves by sublimation inhigh'vacuo and so on in somecases itseems to be commendable to prepare first'the addition compoundswith ketone reagents. Substances giving colored addition compounds as.e. g., nitro-phenvlhydrazines are particularly suitable reagents becausethe reaction-mixture thus obtained can be dividedby means of thechromatogra phical, adsorption method by 'I'swett -(see:=-Berichte d.deutschm botan. Gesellschaft V01. 24 (190.6) 9888 284, iurther, themonograph oi Palmer: Carotinoids and a related pigments. New York: 'liheChemical Cat- Co. (1922).) in fractions containing a e.

-. hormone active component and a corpus iutemn hormone activecomponent. alter liberating the adsorbed compounds byeluting liquids e.g., alcohols, ether, chloroform, acetone, diox- I ane, and splitting onthe ketone reagents. the

I pure substances may be obtained by"the' above:

'mentionedmethods oi. disullationiand sublima-- tion in highvacuoandinsome b'y'recrys tallisation. The workingup by means of reagents givingcolored compounds in'connection with the chromatographicaladsorption-method iollowinaj afterwards may also be applied when the.-.in-' itial material is not yet or only partly removed. In these casesseveral. colored adsorption sones may be stated according towhich'unchanged initial material and other ineflicient reactioncomponents may be separated irom the active'sulh.

stances and in some cases alsothe male acting rrom the corpusluteumhormone acting substance. I

The decomposition of the addition is carried .out in the usual er bydilute acids.

particularly by dilute oxalic acid, whereas the I subsequentpurification of the product thus obtained is eilected for instancebysublimation in high vacuo as above described.

- Example .l. .-A solution or 40 'g. cholestenone s. in 2000 cc; glacialacetic acid is heated ln.a

round-bottomed flask at about 50"; 0. Into this solution a solution of67 g. chromic acid in a mixture of 16000. glacial acetic and,% co. wateris allowed to flow in slowly by stirring the whole mixture. .After .theoxidation en gexcess. oi-

chromic acid, 1: any. is destroyed by addition of methanol and theglacial acetic-acid is distilled ofl in vacuc: the-remaining reactionmixture'is taken up with water and shakenseveral with ether. Theetheralextract is washed with water. with a dilute solution'oi causticsoda and oncemore with water; the ether is then 7 lremoved bydistillation. Theresidue is now jireed from volatile substances by adistillation I with water vapors and the remainder shaken out withether. The etheral solution is dried with 1. sodium sulfate [and ireedfrom ether by distillation. The remaining product isjabrownish oil..-*soluble in alcohols, chloroform. ether. benzene, li

groin, sesame oiLslightly soluble in water, having the activity of amale sexual hormone and of 'a l q corpus luteum hormone. The amount 02the hor-; monal'activity depends on the quantity of inactive byproductsparticularly of unconverted in- 1 itial material; As a rule the combcomb unit 1 according to Fuss-cancer (see: "Med. und chem.-..Abhandlungen der med. chem..For'schungsstat-. "ten der 1. G.Farbenindustrie. 701. 2, (1934) pages 194-204) is contained in about 150300 the seminal vesicle unit according to Loewe und I "Voss (see:Klinische Wochenschrift", vol. -9,

page 481) in about 0.75-15 mg .the corpus lute-l. um hormone unit(tested on therabbit; z

Corner and Allen, "Amer. Journ. PhysloL, vol.

'88,"page 326) about 150-300 mg. Products ruff less and sometimes ofmore activity be obtained.

By removing the inactive byproducts especially v the still existingcholestenone the activities can attains five 101d, a ten fold and even ahigher 00].; value and they maybe approximated to the values of puremale sexual hormone substances and pure corpus luteum hormone To attainthis purification, several methods may be applied. For instance theproduct is allowed to-cool in-a refrigerator, whereby the main part orthe, initial material iorms crystals which I may be'separated by suckingthem on. The iil- .trate is diluted with alcohoiy an alcoholic solutionoi semicai-bazid'e acetate is added and is heated on the'water bathior Ihour. 'lhe 1 precipitating mixture. es i= sentialiy -or entirely oi thesemicarbazone oi the still existing cholestenone and of semicarbazonesoithe active substances is separated-by fractional tion. .The activeiretones maybe chtained from the .semicarbazones by decomposing thelatter'by means oi dilute acids as for imtance by dilute oxalic'acid. Byfractional sublimation or other suitable methods they may be separatedin a fraction having the. activity of a male sex- 'ual hormone andair-action of a corpus luteum hormone.

Instead oi purifying by means oi. ketone 5 agents. a further treatmentmay also be carried out byadsorption methods while the main part 7 ofunchanged cholestenone has been removed by refrigerating. 0n applyingalumina or bleaching clay. inactive substances particularlythe initialmaterial are not adsorbed or only in a feeble mannerso that they can beremoved by washing out with the same solvent in which the i-product isdissolved whilst the active substances remain adsorbed and canbe'removed by special. 4'.-

washing liquids. The mixture of substances dissolved in benzene,toluene, xylene or a similar solvent is for instance adsorbed on acolumn of alumina of loose texture filled, e. g., in a wideglass tube.Benzene or one of the other mentioned solvents is sucked through theadsorption: T mixture until the filtrate substantially leaves onevaporating no residue of inactive substances. .The active substancesare now liberated from the adsorbent by suitable eluting liquids as forinstance alcohols. other.

Example 5.-40 I 1000 cc. glacial acetic acid are added to a solution I1o solvent and divided in fractions by the chromatographical adsorptionmethod. To this end the solution, containing the hydraaones is adsorbedon a column oi bleaching clay and separated by developing intodiil'erent colored zones. By

1 sectioning the column according to the diil'erent colored zones theinactive substances can be separated from the active substances and insome casaalsothesubstanceactingasamaiesexual hormone from the substanceacting as a corpus b luteum hormone. The several parts of the column arethen treated with suitable eluents. the substances thus liberateddecomposed by a'dilute acid and further treated. for instance bysublimation in high vacuo I and fractional above men- Example 2. A'-

of 40 "g. cholestenone in 2000 cc. glacial acetic acid is heated in around bottomed flask at about30'. C. Into this solution to are droppedby stirring it 3600 g. of a 5% aqueous solution of potassiumpermanganate and 750 g. sulfuric acideach solution by itself or bothoithem 'asamixture. Alter theoxidaticn theresction-mixtureistreated'asdescribed in Ex- 8 ample l. 1

Example 3.- -A- solution of 40 g. cholestenone in 2000 cc. glacialacetic acid is treated at 40-50 C. by constant stirring with a solutionof 216 g. acetate or tetravalent lead in 800 cc. glacial acetic Aw acid.The oxidation mixture thus obtained is workedupasdescribedins'lxamplc l.v Example 4.--A solution of .40 g. cholestenone in 1000 cc. benzene, isshaken in a bottle for several daysat a room temperature with 1600 cc.a; of a 5% solution of potassium permanganate and '350 cc.-20% sulfuricacid. the dioxide of manganese thus formed is reduced by simurous acidand the reaction mixture worked "up-a3 described in Example 1. I g.cholestenone dissolved in oi 200 g. chromic acid in 100 cc. water and900 cc. glacial acetic acid in such a manner that the two solutions dropsubstantially at the same time I I on a rotating glass rod which causesthemtoreact andto flow directly into cold water. By this method aparticularly short reaction time is obtamed.

00 ii any, is removed by means oi formic aldehyde;

the considerably diluted solution is now evaporated and the remainder isfurther treated as described in Example 1. Various changes may bemade inthe details 0 disclosed in the foregoing specification without tion,orfsacriilcing the departing from the luv advantages thereof. 1;

In the claims ailixed to this-specification no selection of anyparticular modification of the I invention is intended to the exclusion01 other modifications thereof and therefore nolimitations are intendedother than those imposed by t the claims. We claim:

u 1. The process of producing hormone use subsubstance further treatedas above de-.

- containing large amounts Aiter the oxidation,

When the two solutions have entirely reacted together, an excess of theoxidizing agent.

oneness 3 stances having the activity of a male sexual hormone as wellas of a corpus luteum hormone which consists of treating a mixturecontaining cholestenone by oxidizing agents consisting of the groupincluding oxygen containing large amounts of available oxygen, compoundsof tetravalent lead and acids derived from hydrogen peroxide.

2. The process of producing hormone like substances having the activityof a male sexual horl0 mone as well as of a corpus luteum hormone whichconsists of treating-a mixture containing cholestenone by oxidizingagents consisting of the group including oxy en compounds of metalscontaining large amo ts of available oxygen, ll compounds of tetravalentlead and acids derived from hydrogen peroxide and separating theproduced active substances from the reaction mix- 3. The process ofproducing hormone like substances having the activity of a male sexualhormone as well as of a corpus luteum hormone which consists of treatingcholestenone by oxidizing agents consisting of the group includingoxygen compounds oi metals containing large amounts oi available oxygen,compounds of tetravalent lead and acids derived from hydrogen peroxideand separating the produced active substances irom the'reaction mixture.

4. The process according to claim 1 of producing hormone like substanceshaving the activity or a male sexual hormone as well as 01 a corpusluteum hormone which consists of carrying out the oxidation by means orchromic acid.

5. The process of producing hormone like sub-' 5 stances having theactivity of a male sexual hormone as well as of a corpus luteum hormonewhich consists of treating a mixture containing cholestenone byoxidizing agents consisting of the group including oxygen compounds oi?metals Q oi available oxygen, compolmds of tetravalent lead and acidsvderived from hydrogen peroxide, removing from the reaction mixture theacid components by neutralising agents, the volatile components bydistila latlon andseparating the produced active substances from thereaction mixture.

6. The process of producing hormone like substances having the activityoi a male sexual hormone as well asof a corpus luteum hormone on whichconsists oi treating a mixture containing cholestenone by oxidizingagents consisting of the group including oxygen compounds oi metalscontaining large amounts of available oxygen,

compounds 01 tetravalent lead and acids derived as I from hydrogenperoxide. removing from the reaction mixture the acid components byneutralising agents. the volatile components by distillation andseparating the produced active substances from the reaction mixture byfractional so.

crystallisation at low temperatures.

7. The process of producing hormone like substances having the activityoi a male sexual hormone' as wellas 0! a corpus luteum hormone whichconsists of treating a mixture containing 05 group including oxygencompounds oi metals containing large amounts of available oxygen,compounds or tetravalent lead and acids derived from hydrogen peroxide,removing from the reaction 70 mixture the acid components byneutralising agents, the volatile components by distillation andseparating the produced active substances from the reaction mixture byadsorption agents.

8. The process of producing hormone like sub- 1;

compounds 01' metals 6 stances having the activity of a male sexualhormone as well as of a corpus luteum hormone which consists of treatinga mixture containing cholestenone by oxidizing agents consisting of thegroup including oxygen compounds of metals containing large amounts ofavailable oxygen, compounds of tetravalent lead and acids derived fromhydrogen peroxide, removing from the reaction mixture the acidcomponents by neutralising agents, the volatile components bydistillation and treating the product thus obtained with ketonereagents.

n 9. The process oi. producing hormone like substances having theactivity of a male sexual hormone as well as of a corpus luteum hormonewhich consists of treating a mixture containing cholestenone byoxidizing agents consisting of the group including oiwgen compounds ofmetals containing large amounts of available oxygen, com- U pounds oftetravalent lead and acids derived from hydrogen peroxide, removing fromthe reaction mixture the acid components by neutralising agents, thevolatile components by distillation and treating the product thusobtained with ketone reagents, and separating the formed additionproducts by fractional crystallisation from the reaction mixture.

10. A new hormone product, an oxidation product o1 cholestenone, abrownish oil of ketone.

11. A hormone-like substance having the activity of a male sexualhormone and of a corpus iuteum hormone derived from substancescontaining cholestenone by oxidizing agents consisting of the groupincluding oxygen compounds or metals containing large amounts ofavailable oxygen, compounds of tetravalent lead and acids derived fromhydrogen peroxide.

12. A hormone-like substance having the activity of a male sexualhormone and o! a corpus luteum hormone derived from substancescontaining choiestenone by chromic acid.

13. A hormone-like substance having the activity of a male sexualhormone and of a corpus luteum hormone derived from substancescontaining cholestenone by permanganic acid.

wrmmmv: nmscrman FRITZ mnuscn.

